Use of schizofia schizofiae in preparing a drug for preventing or treating non-alcoholic fatty hepatitis

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a use of schizoflavin in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease. Experiments have confirmed that the compound of the present invention can significantly improve the lipid accumulation and liver fibrosis pathological state of mice induced by a choline-deficient L-amino acid high-fat diet (CDAHFD) after oral administration, significantly reduce the levels of AST, ALT, liver hydroxyproline and TG in serum, and significantly downregulate the expression of fibrosis-related genes and inflammation-related genes in liver tissue. The compound is further expected to be developed as a drug for preventing or treating non-alcoholic liver disease. The structural formula of the compound is shown in Formula I. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to use of schizofiarin in preparing a drug for preventing or treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury that is closely related to insulin resistance and genetic susceptibility. Non-alcoholic steatohepatitis (NASH) is a malignant form of NAFLD, characterized by fat accumulation in the liver, accompanied by inflammation and fibrosis. NASH and hepatocellular fibrosis have no obvious clinical symptoms in the early stages. When obvious symptoms appear, they have basically developed into mid-to-late stage liver cirrhosis.

[0003] The "multiple hits" pathogenic mechanism of NASH is complicated and has not been clearly elucidated so far, making the treatment strategy of NASH difficult. At present, the treatment of NASH is through lifestyle changes, weight loss surgery or liver protection drug-assisted therapy. At present, there are no new therapeutic drugs for NASH approved for marketing in China. Due to the complexity of the pathogenesis of human NASH, there is currently no single animal model that can reflect all the symptoms of human NASH. The diet-induced model is currently considered to be the model that is closest to the clinical progression of NASH patients. Among them, the choline-deficient L-amino acid high-fat diet ( C holine- D eficient,L- A mino acid-defined, H igh-Fat D The CDAHFD (iet, CDAHFD) model can better reflect the histological characteristics of human NASH and can cause more severe inflammation, oxidative stress, apoptosis and fibrosis.

[0004] Diphyllin is a natural product of aromatic naphtholide lignans widely distributed in the plant kingdom. It has a wide range of biological activities, including antibacterial, antioxidant, liver protection, antiviral, platelet activation factor inhibition and anti-tumor activities. In terms of metabolic regulation, the patent (ZL202010051687.7) has reported its role in regulating blood sugar, and the paper (Front. Endocrinol. 2020, 11: 592818) reported that it can improve high-fat diet-induced obesity by regulating brown and beige adipocytes. Currently, no studies have mentioned its application in improving NASH. Summary of the invention

[0005] The purpose of the present invention is to provide a use of schizoflavone in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease. It is reported for the first time that schizoflavone has the ability to improve lipid accumulation and liver fibrosis pathological state in mice induced by a choline-deficient L-amino acid high-fat diet (CDAHFD), significantly reduce serum AST, ALT and liver hydroxyproline and TG levels, and significantly downregulate the expression of fibrosis-related genes and inflammation-related genes in liver tissue.

[0006] The specific technical solutions of the present invention are as follows:

[0007] A use of diphyllin or a pharmaceutically acceptable salt thereof in preparing a drug for preventing or treating non-alcoholic hepatitis, wherein the structural formula of diphyllin is as shown in Formula I:

[0008]

[0009] By adopting the above technical scheme: The present invention evaluates the effect of diphyllin (Formula I) on CDAHFD-induced NASH in C57BL / 6 mice after oral administration (100 mg / kg / day) for 8 consecutive weeks. The experimental results show that the compound can significantly improve the pathological state of lipid accumulation and liver fibrosis in CDAHFD-induced mice after oral administration, significantly reduce the levels of AST, ALT, liver hydroxyproline and TG in serum, and significantly downregulate the expression of fibrosis-related genes (αSMA, Col1a, Col3a, Col4a) and inflammation-related genes (TGFβ, IL-1β, TNF-α) in liver tissue, indicating that it has the effect of improving NASH.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention reports for the first time that schizoflavin has the ability to improve lipid accumulation and liver fibrosis pathological state in mice induced by choline-deficient L-amino acid high-fat diet (CDAHFD), significantly reduce serum AST, ALT, liver hydroxyproline and TG levels, and significantly downregulate the expression of fibrosis-related genes and inflammation-related genes in liver tissue. Therefore, the present invention has the potential value of being developed as a NASH drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1Schematic diagram of the improvement of CDAHFD-induced NASH in C57BL / 6 mice (n=6-8) by diphyllin in the present invention. (A) Oil red and Sirius red staining of liver sections of mice in the Vehicle group (CDAHFD group) and diphyllin group (CDAHFD + oral administration of 100 mg / kg / d) after 8 weeks; (B, C) ALT and AST levels in serum of mice in the Vehicle group, diphyllin group, and NC group (normal diet group) after 8 weeks of feeding; (D, E) Liver hydroxyproline and TG levels in the liver of mice in the Vehicle group and diphyllin group after 8 weeks of feeding; (F) Expression of fibrosis-related genes and inflammation-related genes in the liver of mice in the Vehicle group and diphyllin group after 8 weeks of feeding. DETAILED DESCRIPTION

[0013] In order to facilitate the understanding of the technical solution of the present invention, a detailed description is given below in conjunction with the accompanying drawings and specific embodiments.

[0014] The terms used in the present invention, unless otherwise specified, generally have the meanings that are commonly understood by those of ordinary skill in the art.

[0015] The present invention is further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only intended to illustrate the present invention, and are not intended to limit the scope of the present invention in any way.

[0016] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0017] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0018] Example 1

[0019] Preparation of compound I of the present invention:

[0020]

[0021] S1 (33.2 g, 200 mmol) was dissolved in methanol (160 mL), and Br was slowly added dropwise under ice bath. 2 (11.26mL, 220mmol), after the dropwise addition, stirring was continued at room temperature for 6 hours. After TLC detection, 300mL of ice water was added to precipitate a large amount of solid, and the pH was adjusted to 9 with 2mol / L sodium hydroxide aqueous solution, filtered, and the aluminum cake was washed with ice water and dried to obtain 49.1g of white solid S2, with a yield of 95%. 1H NMR (400MHz, Chloroform-d) δ10.18(s,1H),7.41(s,1H),7.05(s,1H),3.96(s,3H),3.92(s,3H).

[0022] S2 (9.76 g, 40 mmol), ethylene glycol (3.35 mL, 60 mmol), and p-toluenesulfonic acid (480 mg) were refluxed in toluene (200 mL). The water was separated using a Dean-Stark water separator. The reaction was detected by TLC (3 h). Triethylamine (10 mL) was added and stirred for 15 min. The oil was concentrated under reduced pressure to obtain an oil. Ethyl acetate (200 mL) was added to dilute the oil, and the oil was washed with 2 mol / L aqueous sodium hydroxide solution (2×100 mL). The oil was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a white solid. The solid was slurried with ethanol and washed with hot ethanol to obtain 10.2 g of white solid S3 with a yield of 89%. 1 H NMR (400MHz, Chloroform-d) δ7.11(s,1H),7.01(s,1H),5.99(s,1H),4.17(m,2H),4.06(m,2H),3.89(s,3H),3.88(s,3H).

[0023] S3 (2.9 g, 10 mmol) was placed in a two-necked bottle, vacuumed, replaced with nitrogen, 40 mL of ultra-dry tetrahydrofuran was injected, butyl lithium (2.5 mol / L, 4.8 mL, 12 mmol) was slowly added dropwise at -78 ° C, stirred for 15 min, and a solution of S4 piperonal (1.5 g, 10 mmol) in ultra-dry tetrahydrofuran (10 mL) was added dropwise, and continued to stir for 30 min, gradually returned to room temperature, and stirred for another 2.5 hours. After the reaction was completed, 50 mL of glacial acetic acid and diethyl butynedioate (1.7 g, 10 mmol) were added, and heated to reflux at 140 ° C for one hour. The tetrahydrofuran was removed by rotary evaporation, and the solution pH was adjusted to about 7 with a saturated sodium bicarbonate solution, extracted with dichloromethane, and column chromatography, P / E = 3:1, to obtain 3.01 g of yellow solid S5, with a yield of 64%. S5 (3.01 g, 6.4 mmol) was dissolved in 40 mL of tetrahydrofuran, covered with a rubber stopper with a balloon, and borane dimethyl sulfide complex (6.4 mL, 64 mmol) was added dropwise by injection, and reacted overnight. 20 mL of methanol was used to quench the reaction, tetrahydrofuran was removed by rotary evaporation, and the pH was adjusted to 2-3 with 2 mol / L hydrochloric acid, stirred for 30 min, extracted with dichloromethane, and 866.4 mg of salviae sutchuenensis (Formula I) was obtained by recrystallization with methanol, with a yield of 36%. 1 H NMR (400 MHz, DMSO-d 6)δ10.40(s,1H),7.62(s,1H),7.02(d,J=7.9Hz,1H),6.96(s,1H),6.86(d,J=1.6Hz,1 H), 6.75 (dd, J = 7.9, 1.76Hz, 1H), 6.11 (s, 2H), 5.36 (s, 2H), 3.94 (s, 3H), 3.65 (s, 3H).

[0024] Example 2

[0025] Compound I of the present invention improves NASH in mice induced by choline-deficient L-amino acid high-fat diet (CDAHFD):

[0026] C57BL / 6 mice (male) were purchased from the animal room of our unit at 4 to 5 weeks of age and fed adaptively for one week. The random body weight of all mice was measured 2 days before administration. According to the random body weight of the mice, the mice were divided into 3 groups, namely Vehicle group (CDAHFD group), diphyllin group (CDAHFD + oral administration of 100 mg / kg / d), and NC group (normal diet group). During the animal experiment, the weight of the mice was weighed and recorded every week. After 8 weeks of continuous feeding, the mice in each group were treated separately, and the blood of the mice was collected by eye removal. The mice were then dissected, the entire liver was removed, and stored in liquid nitrogen for later use.

[0027] Oil Red O staining of liver sections: The thickness of the frozen sections was 4-5 μm. The sections were stained with Oil Red O staining solution (Oil Red O stock solution was mixed with double distilled water in a ratio of 3:2, and then placed in a clean EP tube and allowed to stand for 10 min). The sections were sealed and stained for 10-15 min. The sections were washed in distilled water for 3 times, and sealed with neutral resin after drying for imaging.

[0028] Sirius red staining of liver sections: Sirius red dye was added to the frozen sections to cover the tissue, and the sections were stained for 50 minutes in the dark. The sections were taken out and washed with deionized water, and then sealed with neutral resin after drying for image acquisition.

[0029] The results of liver slice experiments showed that the natural product diphyllin (Formula I) could significantly reduce the liver lipid accumulation and liver fibrosis pathological state induced by CDAHFD in mice (Appendix Figure 1 A).

[0030] Determination of serum biochemical enzymes ALT and AST: Blood was collected from mice using the eyeball removal method. 1.0 mL of blood sample was placed in a 1.5 mL EP centrifuge tube. After standing at room temperature for 1 hour, 200 μL of the supernatant was aspirated into a new EP centrifuge tube and placed at 4°C overnight. Serum ALT and AST levels were determined using the corresponding kits. The experimental results showed that the natural product diphyllin (Formula I) could significantly reduce the AST and ALT levels in the serum of CDAHFD-induced mice (see Appendix). Figure 1 B and 1C), reflecting the effect of improving liver damage.

[0031] Determination of liver hydroxyproline content: Weigh 50 mg of liver tissue, add 200 μL of anhydrous ethanol, homogenize mechanically on ice, and take 2.5 μL of supernatant after centrifugation. Determine the liver hydroxyproline content according to the corresponding kit. Collagen is the main component of the extracellular matrix during liver fibrosis, so the liver tissue hydroxyproline content can objectively reflect the degree of liver fibrosis. The experimental results show that the natural product diphyllin (Formula I) can significantly reduce the liver hydroxyproline content of CDAHFD-induced mice (see Appendix). Figure 1 D), indicating that it has the effect of improving liver fibrosis.

[0032] Determination of liver TG content: Weigh 50 mg of liver tissue, add 200 μL of anhydrous ethanol, homogenize mechanically on ice, and take 2.5 μL of supernatant after centrifugation. Determine the TG content according to the corresponding kit. The experimental results show that the natural product diphyllin (Formula I) can significantly reduce the content of TG in the liver of mice induced by CDAHFD (Appendix Figure 1 E), indicating that it can significantly improve liver lipid accumulation in the mouse NASH model.

[0033] Fluorescence quantitative PCR test method: ① RNA extraction: Take out the frozen livers of each group, smash and grind them into powder in a pre-cooled mortar, quickly transfer them into an EP tube, add 950μL TRIzol reagent, shake and mix thoroughly, add chloroform with 1 / 5 volume of TRIzol reagent, mix thoroughly, let stand for 2-3min until stratification, centrifuge at 4℃ 12000rpm for 15min, draw the upper aqueous phase into a new EP tube, add isopropanol with the same volume of the upper aqueous phase, and place at -20℃ for precipitation overnight. White precipitate can be seen after centrifugation at 4℃ 12000rpm for 10min. Wash the precipitate once with pre-cooled 70% ethanol, centrifuge at 4℃ 12000rpm for 10 minutes, remove the ethanol, open the lid and evaporate at room temperature for 10 minutes, finally add an appropriate volume of RNAase free sterile water to dissolve the RNA and measure the RNA concentration; ② Reverse transcription: According to the instruction system in the PrimeScript Reverse Transcriptase kit, take 1μg RNA for reverse transcription, inactivate it in a water bath at 37℃ for 15min, and in a water bath at 85℃ for 5s, and then dilute the obtained cDNA tenfold; ③ Real-time quantitative PCR: Take 8.8μL of template cDNA, 1.2μL of 5μM forward and reverse primer mixture, and 10μL of 2×SYBR Green qPCR Master Mix (Biotool) to prepare a 20μL system reaction mixture for PCR amplification. The reaction conditions are: 95℃ pre-denaturation for 5min, 95℃ denaturation reaction for 30s, 60℃ annealing for 30s, 72℃ extension for 30s, and the number of reaction cycles is 40. The obtained data and the internal reference gene were normalized. The experimental results are shown in the attached Figure 1 As shown in Figure F, the natural product diphyllin (Formula I) can significantly downregulate the expression of fibrosis-related genes (αSMA, Col1a, Col3a, Col4a) and inflammation-related genes (TGFβ, IL-1β, TNF-α) in liver tissue, indicating that it has the effect of improving NASH.

[0034] In summary, the present invention reports for the first time that schizoflavin has the ability to improve lipid accumulation and liver fibrosis pathological state in mice induced by choline-deficient L-amino acid high-fat diet (CDAHFD), significantly reduce serum AST, ALT, liver hydroxyproline and TG levels, and significantly downregulate the expression of fibrosis-related genes and inflammation-related genes in liver tissue. Therefore, the present invention has the potential value of being developed as a NASH drug.

[0035] The above are only preferred embodiments of the present invention. The protection scope of the present invention shall be based on the scope defined by the claims. Several improvements and modifications made by those skilled in the art without departing from the spirit and scope of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. Use of diphyllin or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating non-alcoholic hepatitis, characterized in that: The structural formula of diphyllin is shown in Formula I:

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